The baculovirus expression system is well known for its use as eukaryotic cloning and expression vector (King, L A & R D Possee, 1992, The baculovirus expression system, Chapman and Hall, UK; O'Reilly, D R et al., 1992. Baculovirus Expression Vectors: A Laboratory Manual. New York: W.H. Freeman). Advantages of the baculovirus expression system are among others that the expressed proteins are almost always soluble, correctly folded and biologically active. Further advantages include high protein expression levels, faster production, suitability for expression of large proteins and suitability for large-scale production. However, in large-scale or continuous production of heterologous proteins using the baculovirus expression system in insect cell bioreactors, the instability of production levels, also known as the passage effect, is a major obstacle. This effect is at least in part due to recombination between repeated homologous sequences in the baculoviral DNA.
The baculovirus expression system has also successfully been used for the production of recombinant adeno-associated virus (AAV) vectors (Urabe et al., 2002, Hum. Gene Ther. 13:1935-43; U.S. Pat. No. 6,723,551 and US 2004/0197895). AAV may be considered as one of the most promising viral vectors for human gene therapy. AAV has the ability to efficiently infect dividing as well as non-dividing human cells, the AAV viral genome integrates into a single chromosomal site in the host cell's genome, and most importantly, even though AAV is present in many humans it has never been associated with any disease. In view of these advantages, recombinant adeno-associated virus (rAAV) is being evaluated in gene therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, hyperlipoproteinemia type I and other diseases.
To overcome problems with mammalian productions systems for AAV (Urabe et al., 2002, supra) developed an AAV production system in insect cells. For production of AAV in insect cells some modifications were necessary in order to achieve the correct stoichiometry of the three AAV capsid proteins (VP1, VP2 and VP3), which relies on a combination of alternate usage of two splice acceptor sites and the suboptimal utilization of an ACG initiation codon for VP2 that is not accurately reproduced by insect cells. To mimic the correct stoichiometry of the capsid proteins in insect cells Urabe et al. (2002, supra) use a construct that is transcribed into a single polycistronic messenger that is able to express all three VP proteins without requiring splicing and wherein the most upstream initiator codon is replaced by the suboptimal initiator codon ACG. WO2007/046703 discloses further improvement of the infectivity of baculovirus-produced rAAV vectors based production by optimisation of the stoichiometry of AAV capsid proteins as produced in insect cells.
For expression of the AAV Rep proteins in the AAV insect cell expression system as initially developed by Urabe et al. (2002, supra), a recombinant baculovirus construct is used that harbours two independent Rep expression units (one for Rep78 and one for Rep52), each under the control of a separate insect cell promoter, the ΔIE1 and PolH promoters, respectively. However, Kohlbrenner et al. (2005, Mol. Ther. 12 1217-25; and WO2005/072364) reported that the baculovirus construct for expression of the two Rep proteins, as used by Urabe et al. (supra) suffers from an inherent instability. By splitting the palindromic orientation of the two Rep genes in Urabe's (supra) original vector and designing two separate baculovirus vectors for expressing Rep52 and Rep78, Kohlbrenner et al. (supra) increased the passaging stability of the vector. However, despite the consistent expression of Rep78 and Rep52 from the two independent baculovirus-Rep constructs in insect cells over at least 5 passages, rAAV vector yield is 5 to 10-fold lower as compared to the original baculovirus-Rep construct designed by Urabe et al. (2002, supra).
In WO2007/148971, the present inventors have significantly improved the stability of rAAV vector production in insect cells by using a single coding sequence for the Rep78 and Rep52 proteins wherein a suboptimal initiator codon is used for the Rep78 protein that is partially skipped by the scanning ribosomes to allow for initiation of translation to also occur further downstream at the initiation codon of the Rep52 protein.
International patent application WO 2007/084773 discloses a method of rAAV production in insect cells, wherein the production of infectious viral particles is increased by supplementing VP1 relative to VP2 and VP3. Supplementation can be effected by introducing into the insect cell a capsid vector comprising nucleotide sequences expressing VP1, VP2 and VP3 and additionally introducing into the insect cell a nucleotide sequences expressing VP1, which may be either on the same capsid vector or on a different vector.
There is however still a need for further improvements in large scale (commercial) production of parvoviral vectors in insect cells. Thus it is an object of the present invention to provide for means and methods that allow for stable and high yield (large scale) production of parvoviral vectors and for production which results in an improved full:empty particle ratio (i.e., a greater proportion of filled particles).